Fertilized egg isolate and uses thereof

ABSTRACT

Fertilized egg isolate, methods for preparing the fertilized egg isolate and uses thereof for treating mental health disorders and disease or conditions mediated by or associated with one or more glutamate receptors or by the neurokinin 2 (NK2) receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 12/812,335, filedSep. 21, 2010, which is a National Stage Entry of PCT/CA09/00019, filedJan. 9, 2009, which claims the benefit of U.S. Provisional ApplicationNo. 61/020,541, filed Jan. 11, 2008 and U.S. Provisional Application No.61/033,184, filed Mar. 3, 2008, which are hereby incorporated byreference.

FIELD OF THE INVENTION

The invention relates to fertilized egg isolate including that preparedby any of the processes set forth in the description and uses thereoffor treating mental health disorders.

BACKGROUND OF THE INVENTION

Major depressive disorder (also known as major depression, clinicaldepression, unipolar depression and unipolar disorder) is very prevalentin the general population. Most recent North American data show a 14.5%lifetime risk of major depression in adults and 8.1% one year prevalence(Results from the 2004 National Survey on Drug Use and Health: Nationalfindings; Revisions as of Sep. 8, 2005; Department of Health and HumanServices. Substance Abuse and Mental Health Services AdministrationOffice of Applied Studies).

The mean duration of a depressive episode with modern treatments isabout 16 weeks, although some data suggest a longer duration of about6-8 months, far less than in the pre-antidepressant therapy era when theduration was about 18 months (Kendler, McLeod, Patten).

Antidepressants have had a very positive impact on the treatment ofdepression and on reducing the suffering of patients. Patients withdepression are often impaired in function and frequently have co-morbiddisorders such as substance abuse that can be attributed to theunderlying depression. Depression leads to increased utilization ofhealth services and can have a devastating impact on social structureand societal economics.

The cause of depression is not fully known. Disturbance of monoaminesynthesis and activity has been a prominent etiological theory ofdepression for the past few decades and support for this has beenstrengthened by the effectiveness of medications that enhance monoamineactivity, particularly those which are serotonergic and/ornoradrenergic. However, any given antidepressant is only effective in asubset of depressed patients and often only partially so. Currenttreatments administered in controlled trials in academic settings withselected samples show efficacy in only about 60% of patients and onlyabout half of these have full remission of symptoms. This is importantsince the presence of residual symptoms is a strong predictor ofrelapse. There are other physiological changes associated withdepression which suggest a more complex interplay of etiological factorsincluding the role of second messengers mediating membrane bound andintracellular processes. This has led to investigation of hormonalpathways such as the hypothalmic-pituitary-adrenal (HPA) axis (theactivity of which is elevated in 20-40% of community-dwelling patientswith depression), thyroid axis (5-10% of patients evaluated withdepression have previously undetected thyroid dysfunction), growthhormone, prolactin and the role of inflammatory processes and theirmarkers such as interleukin 1 and 6 and tumour necrosis factor.

Most persons with major depressive disorder experience some degree ofsymptom return, and 20-30% exhibit a chronic course (defined as asyndromal level of depressive symptom severity for two years or more(Treatment of Chronic Depression (Editorial))).

All depressed persons require continuation of pharmacotherapy to preventrelapse and permit recovery. A substantial proportion of depressedpatients require maintenance pharmacotherapy to prevent recurrence andfurther consolidate psychosocial recovery. However, while one of themajor factors in effective antidepressant therapy is maintaining thepatient on an adequate dose of medication for an adequate duration, thisis often difficult. Many patients fear taking current antidepressantsbecause of real or imagined physical effects. Some patients prefer touse so-called natural health promoting substances andnon-pharmacological interventions. Patients who are prepared to takeantidepressants often encounter a wide array of side effects which leadsthem to be non-compliant or to reject therapy entirely. Selectiveserotonin reuptake inhibitors (SSRI) for example, commonly inducegastrointestinal upset, headaches, sleep disturbance and significantsexual impairments among many other side effects. Most antidepressantshave at least some significant side effects and these limit clinicians'capacity to effectively treat many patients.

Depression can be associated with other disorders and/or syndromes,including anxiety, such as generalized anxiety disorder, sexualdysfunction, seasonal affective disorder, social anxiety disorder,otherwise known as social phobia, bipolar disorder, and dementia.

It is widely acknowledged that the limited efficacy, often unacceptableside effects and physiological factors that may induce or otherwiseaffect the course of depression make it necessary to continue to searchfor new compounds with novel pharmacological actions to address themajor public health problem of depression.

BRIEF DESCRIPTION OF THE FIGURES

The person skilled in the relevant arts will understand that thefigures, described below, are for illustration purposes only. Thefigures are not intended to limit the scope of the invention in any way.

FIG. 1 is an image of an embryo removed from an egg according toembodiments of the present invention.

FIG. 2 is an image of a freeze-dried embryo according to embodiments ofthe present invention.

FIG. 3 is an image of freeze-dried embryos according to embodiments ofthe present invention.

FIG. 4 is an image of pulverized freeze-dried embryo according toembodiments of the present invention.

FIG. 5 is an image of a slurry of a number of embryos according toembodiments of the present invention.

FIG. 6 shows an HPLC chromatogram of a fertilized egg isolate accordingto embodiments of the present invention.

FIG. 7 shows the results of analyses of a fertilized egg isolateaccording to embodiments of the present invention.

FIG. 8 shows a graph of the effect of various concentrations of thefertilized egg isolate Sample #20 Top Isolate (μg/mL) on binding ofradio-labeled CGP 39653 to the agonist site (ionotropic) of the NMDAglutamate receptor (measured as percent of specific binding) as well asthe IC₅₀ and K_(i) for NMDA and Sample #20 Top Isolate.

FIG. 9 shows a graph of the effect of various concentrations of thefertilized egg isolate Sample #20 Top Isolate (μg/mL) on binding ofradio-labeled kainic acid to the kainate site (ionotropic) of thekainate glutamate receptor (measured as percent of specific binding) aswell as the IC₅₀ and K_(i), for kainic acid and Sample #20 Top Isolate.

FIG. 10 shows a graph of the effect of various concentrations of thefertilized egg isolate Sample #20 Top Isolate (μg/mL) on binding ofradio-labeled α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)to the AMPA site (ionotropic) of the AMPA receptor (measured as percentof specific binding) as well as the IC₅₀ and K_(i) for (+/−) AMPA HBrand Sample #20 Top Isolate.

FIG. 11 shows a graph of the effect of various concentrations of thefertilized egg isolate Sample #20 Top Isolate (μg/mL) on binding ofradio-labeled MDL-105,519 to the glycine site that isstrychnine-insensitive (ionotropic) of the NMDA glutamate receptor(measured as percent of specific binding) as well as the IC₅₀ and K_(i)for MBL-105,519 and Sample #20 Top Isolate.

FIG. 12 shows a graph of the effect of various concentrations of thefertilized egg isolate Sample #20 Top Isolate (μg/mL) on binding ofneurokinin A (NKA) to the NK2 receptor (measured as percent of specificbinding) as well as the IC₅₀ and K_(i) for neurokinin A and Sample #20Top Isolate.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, fertilized egg isolates anduses thereof to treat mental health disorders are described.

Fertilized Egg Isolates—Preparation

In accordance with an aspect of the invention, a process for preparing apowdered sample of at least one embryo or preparing a powdered samplefrom a portion or all of the contents of a fertilized egg is provided.At least one fertilized egg is incubated for anywhere from about 3 toabout 15 days, more preferably about 3 to about 5 days, or morepreferably about 6 to about 12 days, and even more preferably about 7 toabout 9 days, from the day the egg is fertilized. Generally speaking,the fertilized egg is incubated for a period of time that allowsangiogenesis to initiate and/or the embryo to mature to the point thatembryos are visible to the naked eye. The eggs can be from a variety oforigins, for example, avian, reptilian, or from egg-laying mammals.Generally speaking, any egg from which an embryo or blood vesselsassociated with an embryo can be removed can be suitable. The eggs arepreferably avian eggs, and can be obtained from any bird that has beenbred for egg production, such as chicken, geese, ducks, and the like.Chicken eggs are preferred for reasons including their availability andability to be mass produced. Incubation can occur in any environment, solong as the eggs are kept at a temperature for extended periods of timethat allows maturation of the embryo. Suitable temperatures forincubation are in the range of about 20° C. to about 60° C., morepreferably in the range of about 25° C. to about 55° C., and morepreferably in the range of about 35° C. to about 45° C. Once the eggsare incubated for a period of time, they are optionally treated toreduce external microflora or otherwise sterilized by any suitablemeans, such as washing the egg shells with a solvent such as ethanol,for example, an about 50% to about 95% solution of ethanol, withsubsequent time allowed to allow evaporation or drying of the solvent,or by rotating the eggs under an ultraviolet (UV) light source for asuitable period of time. Any solvent is preferably evaporated beforefurther manipulation of the egg. The eggs are then cracked to access theinner contents. The eggs can be cracked under aseptic conditions eithermanually or using a suitable mechanical device. This procedure and/orall or most of the procedures described above and below can be conductedin a cooled atmosphere, such as an atmosphere of about 5° C.

According to one aspect of the invention, the contents of the egg arecollected in a container, such as a stainless steel container, which ispreferably sterilized and/or chilled. The contents from the container orfrom the egg can optionally be subjected to a filtration process, forexample, by being placed on a mesh. The mesh openings can be about 0.5to about 4 millimeter, more preferably about 1 millimeter. The mesh ispreferably sterile.

Optionally, the contents of the egg and/or some or all of the brokenshell can be placed directly on the mesh. The contents of the egg and/orsome or all of the broken shell are allowed to filter on the mesh for aperiod of time such that there is substantially no further dripping offluid through the mesh. The broken shell can be removed from thecontents of the egg before, during, or after the filtration process.After the filtration, the solid or solid and semi-solid retentate cancomprise the embryo, vascular connective tissue, a substantial portionor all of the albumen, a substantial portion or all of the chalaza, andthe clear sac. Semi-solid retentate can comprise solid material as wellas a viscous material, such as a gelatinous material, for example,albumen. The retentate or semi-solid retentate can be optionally washedat least once with a suitable solvent, such as a buffer solution,sterile deionized water, or any suitable saline solution. For example,sterile phosphate buffer saline (PBS) can be used.

The retentate can be collected from one egg and then freeze-driedaccording to the processes described herein, or the retentate can becollected from one or more eggs together, and then freeze-driedaccording to the processes described herein.

According to another aspect of the invention, the white albumen portionand/or embryo can be substantially separated from the rest of thecontents of the egg. The white albumen portion may be substantiallyseparated from the rest of the contents by any suitable means, such asdecantation of the white albumen portion, or by suction. The embryo canbe substantially separated from the white albumen portion manually orother suitable means as determined by the skilled person. FIG. 1 showsan example of an embryo that was removed from an egg and washed withbuffer. It will be recognized by those skilled in the art that theembryo can be substantially separated from the white albumen portion andthe rest of the inner contents at the same time. For example, the embryocan be manually removed from the white albumen portion and rest of theinner contents using tweezers or other suitable instrument. In somecases, the embryo can be manually peeled off the yolk sac, which formspart of the rest of the inner contents.

Once the embryo is substantially separated from the white albumenportion and the rest of the inner contents of the egg, the embryo isoptionally washed at least once with a suitable solvent, such as abuffer solution, sterile deionized water, or any suitable salinesolution. For example, sterile phosphate buffer saline (PBS) can beused.

It will be understood for the following methods that reference tocontents of the egg may actually be a reference to the retentate if thecontents have been subjected to a filtration process. It will also beunderstood that a whole fertilized egg can be cracked, the shellremoved, and the whole of the shelled egg frozen and freeze-driedaccording to any of the procedures described above and below, to producea fertilized egg isolate of the present invention. Also, more than onewhole fertilized egg can be cracked, shells removed, the whole of theshelled fertilized eggs combined and blended into a slurry, and frozenand freeze-dried according to any of the procedures described above andbelow.

The contents of the eggs or the embryos are placed in at least onefreezable container. The container can be, for example, a test tube,Petri dish, beaker, stainless steel tray, or plastic container. It ispreferred that the contents or embryos are frozen very soon after beingremoved from the shell, such as within about 2 hours, more preferablywithin about 1 hour, and even more preferably within about 0.5 hours, oras soon as possible. Depending on how long the contents or embryos areto be frozen, the freezing temperature should be in the range of about−50° C. to about 10° C., more preferably in the range of about −40° C.to about 5° C., and even more preferably in the range of about −35° C.to about −25° C. It is preferred that the contents or embryos are frozenfor at least about 6 hours, more preferably at least about 12 hours,even more preferably at least about 24 hours. The frozen contents orembryos may be freeze-dried or lyophilized after a period of time. Thecontents or embryos can be completely frozen before thefreeze-drying/lyophilizing step. FIGS. 2 and 3 show examples of embryosafter they have been freeze-dried.

Optionally, frozen or unfrozen contents or embryos can be pooled in asuitable container, such as a beaker, or a plastic container, and mixedor blended with a suitable solvent, if necessary, to form a slurry. Thesolvent can be suitably aqueous to wet the mixed contents or embryos andbe able to be frozen in a standard laboratory freezer. Suitable solventsinclude water, aqueous buffer, and the like. To form the slurry, it ispreferred that the contents and/or embryos are blended. The contents orembryos can be blended or homogenized with, for example, a hand-heldblender or other suitable means. The slurry can then be frozen asdescribed above and freeze-dried. Freeze-drying is preferably performedat an ultimate temperature in the range of about −80° C. to about −10°C., more preferably in the range of about −65° C. to about −15° C., andeven more preferably in the range of about −40° C. to about −20° C. anda pressure of about 500 millitorr, or other suitable pressure as can bedetermined by the skilled person. The freeze-drying process ispreferably maintained at the ultimate temperature for a period of in therange of about 1 to about 6 hours, more preferably in the range of about2 to about 5 hours, and even more preferably in the range of about 3 toabout 4 hours. The whole freeze-drying process is typically conductedfor a period in the range of about 15 to about 45 hours, more typicallyin the range of about 25 to about 35 hours, and even more typically inthe range of about 28 to about 32 hours.

In accordance with another aspect of the invention, the freeze-driedcontents, freeze-dried embryo, or freeze-dried slurry is dispersedand/or pulverized if necessary to form a substantially homogeneouspowder. The contents that were freeze-dried individually or in smallergroups can be combined together before or after the pulverization stepto form a substantially homogeneous powder. The pulverization can bedone, for example, mechanically using a suitable machine, such as acoffee bean grinder or a hammer mill, or manually using a suitable tool,such as a glass rod. FIG. 4 shows an example of pulverized freeze-driedembryo. The powder is optionally sterilized before being stored and/orused. A suitable sterilization should be one that does not adverselyaffect certain freeze-dried components.

In association with any process described herein, preservatives tocontrol microbial growth can be blended into the powder or concentratebefore it is stored. Preservatives can also be added at another stage ofthe manufacture, including before the freeze-drying or concentrationstep instead of, or in addition to, being added to the powder orconcentrate. Suitable preservatives include common food preservativessuch as 0.5% w/w sodium benzoate and 0.2% w/w potassium sorbate. Othersuitable preservatives could be selected by the skilled person.

The powders produced by the processes disclosed herein can be stored insuitable, substantially air-tight containers. Suitable containersinclude plastic bags, barrels, plastic containers, bottles, combinationsthereof, and the like. For example, the powder can be packaged undercontrolled, aseptic conditions into sterile polyethylene/polypropylenebottles with tamper-proof security seals. The powder can be stored undera substantially dry, inert gas, such as nitrogen. It is preferred thatthe powder be stored at room temperature or cooler, for example, at atemperature in the range of about 10° C. to about 25° C., morepreferably in the range of about 15° C. to about −20° C. For long termstorage, it is preferred that the powder is stored at a temperature ofabout −10° C. or below, or, more preferably, −20° C. or below. Thepowder can be stored for a period of time in a substantially desiccatedatmosphere. The powder can also be vacuum-packed.

In accordance with another embodiment of the present invention, a slurryis prepared as described above, or can be prepared by separating thecontents or embryos of at least one fertilized egg from the egg shell,and pooling the separated contents or embryos in a suitable container.The separated contents or embryos can be cooled during this step. Forexample, the container can be placed on ice to facilitate cooling. Thecontents or embryos can be blended by methods described above to producea slurry. FIG. 5 shows an example of a container of a slurry of embryos.The slurry can be freeze-dried as described above, or partially orwholly used for extraction procedures as follows.

An aqueous extraction procedure can be performed by mixing the slurrywith an aqueous solution for a period of time. The aqueous solution maycomprise water, an aqueous buffer, or any other aqueous solvent. If theaqueous solution comprises water, it is preferred that the water isdistilled and, more preferably, also deionized before use. For example,the water can be treated using reverse osmosis (R.O.). The slurry andthe aqueous solution can be mixed, for example, by stirring for a periodof time, the period of time being in the range of about 5 to about 60minutes, more preferably in the range of about 10 to about 45 minutes,and even more preferably in the range of about 15 to about 40 minutes.It is desired that the aqueous solution has sufficient exposure to thecontents of the slurry so that any substantially hydrophilic moleculesin the solution are dissolved in the aqueous solution. The aqueoussolution can be of a substantially equal volume to the slurry, butvolumes of 1.5 times, 2 times, or even 3 times the volume of the slurrycan be used. Optionally, the mixture can be warmed slightly during themixing step. After the mixing, the aqueous solution can be substantiallyclarified by substantially removing any solid portions in the mixture bysuitable means such as centrifugation or filtration. The clarifiedaqueous portion can then be frozen and freeze dried to produce a powderthat is optionally sterilized according to methods described herein.

According to another aspect of the invention, the slurry produced by anyof the methods described above can be mixed with a substantiallyhydrophobic solvent. The substantially hydrophobic solvent is preferablychilled. Suitable hydrophobic solvents include, for example, ether,chloroform, hexane, petroleum ether, or acetonitrile. For example,ether, especially diethyl ether, can be used. The slurry is mixed withthe hydrophobic solvent for a period of time as described above. As willbe recognized by a person skilled in the relevant arts, any steps of aprocess using a substantially hydrophobic solvent should be conducted ina fume hood or similar device, and the solvents should be kept away fromopen flames or heat sources. After the mixing period, the solid portionsof the mixture can be substantially removed from the solvent portion bysuitable means such as centrifugation or filtration. The solvent portionwill comprise substantially a hydrophobic solvent portion and may alsocomprise an aqueous portion. The solvent portion can be transferred to aseparating funnel or essentially equivalent device to separate theaqueous portion from the hydrophobic solvent portion. If the top layeris the hydrophobic solvent portion, it can be siphoned off the top orremoved from the separating funnel after the bottom aqueous layer isremoved. Alternatively, the bottom aqueous portion can be frozen,thereby allowing the top ether-based layer to be decanted. The aqueousportion can be extracted a number of times, for example, about 3 times,with the hydrophobic solvent. The hydrophobic solvent can be ofsubstantially equal volume to the aqueous portion, or can be 1.5 times,2 times or even 3 times the volume of the aqueous solvent. Other ratiosmay also be suitable.

After the extraction process, all of the hydrophobic isolates can bepooled and concentrated by a suitable method. The concentrated isolatescan be stored at a temperature below room temperature, such as about 5°C. in a suitable container that is substantially sealed from theatmosphere, such as a sealed vial.

According to another aspect of the invention, a slurry produced by anyof the methods described above can be clarified before an extractionprocedure. Preferred clarification steps include methods of filtration,using such filters as sieves or filter papers or pads. Otherclarification steps can include methods of centrifugation. A filter aid,such as Superflow DE™ can be added to the filtrate produced by thefiltering step before further clarification. Some of the resultantfiltrate can be frozen in suitable containers for freeze-drying. Also,some of the resultant filtrate can be mixed with a hydrophobic solventas described above so that an aqueous layer and a hydrophobic layer areformed. The layers can be separated, concentrated, and stored asdescribed herein.

The fertilized egg isolate prepared by various processes describedherein could be formulated to increase potency by repeating and/orcombining processes. For example, aqueous and/or hydrophobic solventextraction can be repeated on the same sample to concentrate activecompounds.

Fertilized Egg Isolate—Uses

Fertilized egg isolate as prepared by the processes described herein, oras prepared by similar processes which will be readily apparent to theskilled person upon learning of the present invention, can be used totreat patients suffering from mental health disorders, includingdepressive mood disorders, such as major depressive disorder, dysthymicdisorder, depressive phase of bipolar disorder, depression due to ageneral medical condition such as depression associated with dementia orschizoaffective disorder, substance-induced depression and seasonalaffective disorder, anxiety disorders, such as generalized anxietydisorder, social phobia and panic disorder, and sexual dysfunction. Aswill be appreciated by those skilled in the relevant arts, treating suchdisorders as depression or anxiety can be a useful approach for treatingother disorders and/or syndromes with which they may be associated, suchas those listed above. In one embodiment, the patient is a human.

As described in detail herein, it has been determined that thefertilized egg isolate of the present invention antagonizes the bindinginteractions of certain ligands with their receptors. In particular, ithas been found that the fertilized egg isolate of the present inventionhas the capacity to displace glutamate from four of its major receptors.In addition, the fertilized egg isolate can displace theneurotransmitter neurokinin A (NKA) from its receptor, neurokinin 2 (NK2receptor). To the knowledge of the present inventors, this is the firstknown instance in which these two receptor groups have been shown to beantagonized by a single substance.

Glutamic acid (glutamate) is one of the most excitatory substanceswithin the human brain. A number of diseases and conditions are known tobe mediated by or associated with activation of one or more glutamatereceptors. These diseases and conditions include depression (see, forexample, Paul, Toro, Mathew 2005, Krystal, Sanacora 2003, Svenningsson,McNally), major depressive disorder (see, for example, Maeng, Chourbaji,Mathew 2008), anxiety (see, for example, Rorick-Kehn), Alzheimer'sdisease (see, for example, Walton, Koch, Hynd), epilepsy (see forexample, Kew, Vincent), schizophrenia (see, for example, McCullumsmith,Lewis, MacDonald, Javitt), impairment of brain cell function afterstroke/ischemia (see, for example, Ginsberg, Kew), amyotrophic lateralsclerosis (Lou Gehrig's Disease) (see, for example, Mathew 2008,Miller), lathyrism (see, for example, Spencer, Ravindranath), autism(see, for example, Chez), mental retardation (see, for example, Bowie),disturbances of cognition (see, for example, Cheon), bipolar depression(see, for example, Mathew 2008) and mania (see, for example, Krystal).Furthermore, it has been shown that antagonists of the glutamatereceptor, such as ketamine and riluzole can be used to treat depression(Mathew 2008), major depressive disorder (Maeng, Mathew 2008, Zarate2004, Sanacora 2007), amyotrophic lateral sclerosis (Mathew 2008,Miller) and bipolar depression (Mathew 2008, Zarate 2005). Theinhibition of activation of one or more glutamate receptors, byinhibiting glutamate's binding to its receptor, can diminish oreliminate conditions or diseases mediated by or associated with one ormore glutamate receptors. Since it has been determined, as describedherein, that the fertilized egg isolate of the present invention bindsto one or more glutamate receptors, the fertilized egg isolate of thepreset invention can be used to treat diseases and conditions associatedwith or mediated by one or more glutamate receptors.

Accordingly, another aspect of the invention features a method fortreating a disease or condition associated with or mediated by aglutamate receptor using fertilized egg isolate, the method comprisingthe step of administering a therapeutically effective amount of thefertilized egg isolate to a patient in need thereof. Among suchglutamate receptors are the ionotropic glutamate receptors, for example,the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor,the kainate receptor and the N-methyl-D-aspartic acid (NMDA) receptor.The disease or condition associated with or mediated by a glutamatereceptor can include depression, major depressive disorder, anxiety,Alzheimer's disease, epilepsy, schizophrenia, impairment of brain cellfunction after stroke/ischemia, amyotrophic lateral sclerosis (LouGehrig's Disease), lathyrism, autism, mental retardation, disturbancesof cognition, bipolar depression or mania.

A number of diseases and conditions are also known to be mediatedthrough or associated with activation of the NK2 receptor. Such diseasesor conditions include depression (see, for example, Dableh, Ahlstedt,Michale, Louis, Steinberg, Salomé, Holmes, Steinberg, Husum), anxiety(see, for example, Ahlstedt, Michale, Louis, Greibel, Steinberg,Stratton, Teixeira, Walsh, Salomé, Holmes), irritable and inflammatorybowel syndrome (see, for example, Ahlstedt, Lecci, Evangelista,Toulouse), inflammatory airway diseases (see, for example, Bai) andurinary incontinence (see, for example, Ahlstedt, Rizzo). Furthermore,it has been shown that antagonists of the NK2 receptor, such assaredutant (SR 48964) can be used to promote antidepressant-like effects(Salomé, Dableh, Steinberg, Michale, Louis) and anxiolytic effects(Teixeira, Salomé, Griebel, Michale, Louis) in animal models, andstudies in humans have also been conducted. The inhibition of activationof the NK2 receptor, by inhibiting NK2's endogenous ligand(s), e.g., NKAfrom binding to its receptor, can diminish or eliminate conditions ordiseases mediated by or associated with the NK2 receptor. Since it hasbeen determined, as described herein, that the fertilized egg isolate ofthe present invention binds to the NK2 receptor, the fertilized eggisolate of the present invention can be used to treat diseases andconditions associated with or mediated by the NK2 receptor.

Accordingly, another aspect of the invention features a method fortreating a disease or condition associated with or mediated by an NK2receptor using fertilized egg isolate, the method comprising the step ofadministering a therapeutically effective amount of the fertilized eggisolate to a patient in need thereof. The diseases or conditionsassociated with or mediated by an NK2 receptor can include depression,anxiety, irritable bowel syndrome or urinary incontinence.

The fertilized egg isolate used in the treatment of a disease orcondition associated with or mediated by one or more of a glutamatereceptor and an NK2 receptor can comprise freeze-dried embryo,ovalbumin, and clear sac from the fertilized egg or it can be producedsuch that it does not comprise a substantial amount of the yolk from thefertilized egg. The fertilized egg isolate can be prepared according tomethods described herein, or by similar methods known to those skilledin the art. In the methods for treating a disease or a conditionassociated with or mediated by a glutamate receptor or an NK2 receptor,the patient may or may not be being treated by psychotherapyconcurrently with the treatment.

Another aspect of the invention is methods of treatment of mental healthdisorders comprising the administration of fertilized egg isolate to apatient in need, and uses of fertilized egg isolate for such purposes.

The fertilized egg isolate described herein can be formulated for andadministered as various dosage forms, such as those adapted foradministration by the oral (including buccal and sublingual), rectal,nasal, topical (including buccal, sublingual, and transdermal), vaginal,rectal, or parenteral (including subcutaneous, intramuscular,intravenous, or intradermal) routes. Particularly preferred are dosageforms adapted for administration by the oral route. Other preferreddosage forms include those adapted for administration by the vaginal orrectal route, such as a suppository.

Fertilized egg isolate formulations adapted for oral administration canbe presented as discrete units such as capsules, tablets,microparticles, powders, granules, solutions or suspensions in aqueousor non-aqueous liquids, edible foams or whips, or oil-in-water emulsionsor water-in-oil emulsions. The fertilized egg isolate can be combinedwith a suitable oral, non-toxic pharmaceutically acceptable inertcarrier. In the case of capsules, a suitable encapsulator, such as agelatin sheath, can be used to encapsulate the fertilized egg isolatealone or in combination with suitable non-toxic pharmaceuticallyacceptable inert carrier(s). In respect of tablets and capsules, forexample, suitable carriers can include, but are not limited to, ediblecarbohydrate, such as, for example, starch or mannitol, flavoring,preservative, dispersants, binders, and coloring agents, and can includefumed silica. The foi mulation can also be prepared to prolong orsustain the release of the fertilized egg isolate, in accordance withmeans known to those skilled in the pharmaceutical formulation arts.

An effective dosage for treatment will depend on the patient. Effectivedosages can range from about 200 to about 6000 mg/day, or from about 500to about 4000 mg/day, or from about 750 mg/day to about 3500 mg/day, orfrom about 800 to 3000 mg/day, or from about 1000 to 2500 mg/day. Forexample, the effective dosage could be about 2000 mg/day.

The term “treat” means improving the disease or condition of a patientto whom the fertilized egg isolate of the present invention is beingadministered. This term includes ameliorating the disease or condition,and such amelioration can be determined using standard tests known inthe art. The term also includes preventing the disease or condition fromoccurring or re-occurring, such as in prophylactic or maintenancetherapy.

Another aspect of the invention features a method for inhibiting anactivity of a glutamate receptor comprising contacting the glutamatereceptor with an effective amount of fertilized egg isolate. This methodcan be performed using an ionotropic glutamate receptor, for example, anAMPA receptor, a kainate receptor or an NMDA receptor.

In addition, the invention features a method for inhibiting an activityof an NK2 receptor comprising contacting the NK2 receptor with aneffective amount of fertilized egg isolate.

The activity of a glutamate receptor or an NK2 receptor can beinhibited, for example, by inhibiting binding of the receptor by itsendogenous ligand(s) (e.g., glutamate for the glutamate receptors andNKA for the NK2 receptor), or commercially available exogenous ligands,such as AMPA, NMDA, kainate, CGP 39653 or MDL-105,509 for the glutamatereceptors, and saredutant for the NK2 receptor. Methods for inhibitingsuch a binding interactions and for detecting such binding inhibitionare known to those skilled in the art, and are also described herein.Activity of the glutamate receptor or NK2 receptor can be inhibiting by100% or by less than 100% (for example, by 90%, 80%, 70%, 60%, 50%, 40%,30%, 20% or 10%). Methods for inhibiting the activity of a glutamatereceptor or an NK2 receptor can be carried out in vitro (for example, ina cell, cell lysate, or a sample containing a portion of a cell, forexample, just the relevant receptor) or in vivo (for example, in a humanpatient).

According to a further embodiment of the invention, the fertilized eggisolate can be formulated with other compounds which may be useful fortreating mental health disorders. For example, the fertilized eggisolate can be formulated with compounds that may inhibit the breakdownof serotonin, such as monoamine oxidase inhibitors.

Still other objects and advantages of the present invention will becomereadily apparent to those skilled in the art from the detaileddescription, wherein only the preferred embodiments are described,simply by way of illustration. As will be realized, the invention iscapable of other and different embodiments, and its several details arecapable of modification in various obvious respects, all withoutdeparting from the invention. Accordingly, the description and examplesbelow are to be regarded as illustrative in nature, and not asrestrictive.

EXAMPLES Example 1

Fertilized hen eggs that were 8-9 days old were selected. The whole eggwas disinfected with 70% ethanol and left in the fume hood to allow the70% ethanol to evaporate. The eggs were broken and the contents weredropped through a sterile 1.0 min mesh. The shells and filtrate werediscarded. The retentate, both solid and liquid, was collected andchilled on ice. The retentate was homogenized at 5° C. The homogenate(slurry) was poured into sterile stainless steel trays, frozen, andfreeze-dried. The dried product was pulverized with a grinder.Preservatives 0.5% w/w sodium benzoate and 0.2% w/w potassium sorbatewere added to the powder, and the mixture was blended. The finishedpowder was stored at 2-8° C. (short term) or −20° C. (long term).

Example 2

Chicken embryos were harvested at 8 days old from fertilized hen eggsaccording to the following method:

-   -   (i) 24 brown eggs were received;    -   (ii) when cracked open, only 22 of 24 eggs where found        fertilized containing the developed embryo (91.6% fertilization        rate);    -   (iii) embryos were removed and washed (×2) briefly in sterile        PBS and immediately frozen at −20° C. in individual 50 mL        (milliliter) test tubes;    -   (iv) 10 test tubes were then freeze dried in a freeze dryer        (shelf temp.=−40° C., condenser temp. −52° C.);    -   (v) at the end of the cycle, the individual embryos (while in        test tube) were crushed with a sterile glass rod and the        resulting powder (1.7 g (grams)) was packaged;    -   (vi) the 12 remaining embryos were pooled in a small beaker and        blended with a small amount of water using a hand held blender;    -   (vii) the resulting slurry was immediately frozen and        subsequently freeze dried as above.

Results

Once dried, the powder (1.7 g) was dispersed and made homogeneous usinga glass rod=Lot# A. Each embryo yielded 0.17 g powder.

The dried slurry (1.6 g) was dispersed and made homogeneous using aglass rod=Lot# B. Each embryo yielded 0.14 g powder (moisture unknown).

Example 3

Sixty fertilized eggs (8 day old embryos) were received. The followingis a description of the preparation of both the aqueous and the etherisolates.

All eggs were sprayed with 70% (v/v) ethanol to sterilize the externalsurface and the eggs were left in the laminar flow hood for the alcoholto evaporate. The eggs were broken and the embryos and the attachedclear sac with its clear watery content were carefully separated fromthe rest of yolk and vascular material. Then, all material, that is, theembryo and clear sacs, was pooled in a beaker that had been chilled onice. Following the harvest, the material was blended with the help of ahand held blender, resulting in a slurry.

The resulting slurry (approx. 200 mL) was split into two equal halves;one was used for extraction with water and the second for etherextraction.

Aqueous Isolate

To the 100 mL slurry, 100 mL of R.O. water was added and the materialwas stirred at room temperature for 30 minutes. The material wasclarified by centrifugation and the aqueous top layer was collected (200mL 1.1% solids). The aqueous top layer was then frozen and subsequentlyfreeze dried. The dry powder weighed 1.0 g.

Ether Isolate

To 100 mL of the slurry, 100 mL of previously chilled ether was added.The mixture was shaken at room temperature and then subjected tocentrifugation. At the end of the centrifugation period (15 minutes at5° C.), the top aqueous/ether layer was transferred to a separatingfunnel and the top ether layer was collected either by siphoning or,after a period of freezing at −20° C. that froze the lower turbidaqueous layer leaving the top clear yellow ether layer unfrozen,decanting.

The lower aqueous layer was extracted again with an equal volume ofether as described above and the process was repeated three times. Theether isolate was then concentrated and dried in the Rotavap™ to a smallvolume (approximately 1 mL). The sample was kept at 5° C.

Example 4

Four hundred twenty fertilized eggs (8-9 day old embryos) were sprayedwith 70% (v/v) ethanol to sterilize the external surface as much aspossible and the eggs were left in a fume hood for the alcohol toevaporate.

The eggs were broken (62 out of 420 were found unfertilized,fertilization rate=85.2%) and the embryos and their attached clear sacwith its clear watery content were carefully separated from the rest ofyolk and vascular material and all material, that is, the embryo andclear sacs, was pooled in a beaker that had been chilled on ice. Theharvested material (approx. 2 L) was kept at 5° C.

Aqueous Isolate

Following the harvest, the material was blended with a hand-held blenderthat produced a slurry. The resulting slurry (approx. 2 L) was passedthrough a metal sieve (MESH no. 20). The filtrate was further clarifiedwith a paper filter pad and DE 6000. The cake left on the filter pad wasdiscarded. To the filtrate, Superflow DE™ was added and the material wasfurther clarified by passing through a filter pad. All filtration wasdone using a Buchner funnel at bench scale level. The majority (about1200-1300 mL) of the resulting filtrate was separated and immediatelyfrozen in trays for freeze drying (1.9% solids). The remaining 500 mLwas mixed with an equal volume of previously chilled ethyl ether in a 1L bottle and kept at 5° C. until separation of the two layers.

Ether Isolate

To 500 mL of the slurry, 500 mL of previously chilled ether was added,the mixture was shaken at room temperature and then subjected tocentrifugation. At the end of the centrifugation period (15 minutes at5° C.) the top aqueous/ether layer was transferred to a separatingfunnel and the top ether layer was collected either by siphoning orafter a period of freezing at −20° C. that will freeze the lower turbidaqueous layer, leaving the top clear yellow ether layer to be decanted.

The lower aqueous layer was extracted again with 300 mL of ether asdescribed above and the isolates were pooled in a round-bottom,previously chilled flask. The ether isolate was then concentrated anddried in the Rotavap™ to a small volume. The sample was kept at 5° C.

Example 5

Fertilized Egg Isolate A

To produce fertilized egg Isolate A, 8-9 day old whole fertilized heneggs were disinfected with 70% ethanol and left in a fume hood to allowthe solvent to evaporate. The eggs were then broken and the contentsdropped on or through a sterile 1.0 mm mesh. The shells and filtratewere discarded. The retentate, which comprised the embryo, clear sac,and all or a substantial part of the albumen and consisted of solid andsemi-solid and/or liquid portions, was chilled on ice and thenhomogenized at 5° C. The homogenate (slurry) was poured into sterilestainless steel trays, and freeze-dried. The dried product waspulverized in a grinder to give Isolate A. To Isolate A, thepreservatives sodium benzoate (0.5% w/w) and potassium sorbate (0.2%w/w) were added and the mixture was blended. The finished powder wasstored at 2-8° C. (short term) or −20° C. (long term).

HPLC Analysis

The finished powder containing fertilized egg Isolate A was analyzed byHigh Performance (or Pressure) Liquid Chromatography (HPLC). The resultswere quantified using a multiple-wave absorption detector. Absorptionwas read at 215 nm. A Pharmacia Superdex 200 10/300GL size exclusioncolumn (10 mm i.d.×300 mm) was used for fractionation. The separationrange of the column was 10 kDa-600 kDa. The column was equilibrated with20 mM phosphate+0.3 M NaCl, pH 7.5. The sample was analyzed at a flowrate of 0.5 mL/min. A representative chromatogram is shown at FIG. 6.

Certificate of Analysis

The finished powder containing Isolate A was also subjected to standardanalytical procedures to measure purity and the content of protein, fat,ash, moisture, and various contaminants. A representative sample of theresults is shown at FIG. 7.

Formulation A Capsules

In order to prepare capsules of Formulation A, 4000.0 g (+/−2%) of thefinished powder containing Isolate A, sodium benzoate (0.5% w/w) andpotassium sorbate (0.2% w/w) was mixed with 40 g (+/−2%) of fumed silicausing geometric dilution. The mixture was sifted, and the mixing andsifting were repeated, resulting in Formulation A. The Formulation Amixture was encapsulated using Mini-Cap 300#0 white capsules to a targetfill weight of 505 mg to produce Formulation A capsules.

Example 6 Study of Formulation a for Treatment of Major DepressiveDisorder (MDD) and Disorders/Symptoms Related Thereto

The efficacy and safety of a fixed dose of Formulation A to treat mentaldisorders, such as MDD and related disorders and symptoms, were studied.This study included evaluation of the effect of Formulation A onreducing symptoms of anxiety, improving quality of life, and improvingsymptoms of sexual dysfunction.

Description of Evaluation Techniques

Hamilton Depression Rating Scale-17 Item—“HAM-D” or “HAM-D 17”

This is a leading rating scale used in North America for evaluatingdepression in a patient. The total scores are interpreted as follows:very severe, >23; severe, 19-22; moderate, 14-18; mild, 8-13; and nodepression, 0-7.

Hamilton Anxiety Rating Scale-14 Item—“HAM-A”

This rating scale evaluates the level of anxiety in a patient. The scorelevels are interpreted as follows: <17, mild; 18-24, mild to moderate;and 25-30, moderate to severe.

Montgomery-Asberg Depression Rating Scale—“MADRS”

This is a leading rating scale used in North America for evaluatingdepression in a patient. The following mean scores correlate with globalseverity measures, according to a study: very severe, 44; severe, 31;moderate, 25; mild, 15; and recovered, 7.

Beck Depression Inventory—“BDI”

This is a commonly employed measure of depressive symptoms typicallyused as a self-assessment instrument. The total score is the simple sumof the 21 item scores. Generally, a score <9 indicates no or minimaldepression, 10-18 indicates mild-to-moderate depression, 19-29 indicatesmoderate-to-severe depression, and >30 indicates severe depression.However, a score of 0-4 may suggest possible denial of depression and ascore of 40-63 may suggest possible exaggeration of depression or ahistrionic or borderline personality disorder.

Arizona Sexual Experience Scale—“ASEX”

This is a 5-item rating scale that quantifies sex drive and evaluateslevels of arousal, vaginal lubrication/penile erection, ability to reachorgasm, and satisfaction from orgasm.

Possible total scores range from 5 to 30, with the higher scoresindicating more sexual dysfunction.

General Health Questionnaire Scoring—“GHQ”

The quality of life dimension may have been assessed with the Short-Form36 (SF-36). This questionnaire evaluates such problems as the ability toconcentrate, feelings of worry, low self-confidence, feelings of lowself-worth, unhappiness, and depression. The scoring is as follows:

-   -   Likert Scale 0, 1, 2, 3 from left to right.    -   12 items, 0 to 3 each item.    -   Score Range 0 to 36.    -   Scores vary by study of population. Scores about 11-12 are        typical.    -   Score >15 evidence of distress.    -   Score >20 suggests severe problems and psychological stress.

Diagnostic and Statistical Manual of Mental Disorders-IV-TextRevision—“DSM-IV TR”

This is the standard diagnostic manual in North America for mentalhealth professionals that comprehensively classifies mental disordersand provides widely accepted criteria for diagnosing them based on thebest empirical evidence available.

The primary effect measured was the repeated analysis of variance withthe score on the HAM-D as the outcome variable. Secondary effectmeasures included the CGI-S and CGI-I, MADRS, SF36, BDI, HAMA and ASEX.

Description of Study

An open-label study was carried out at Mount Sinai Hospital (MSH) inToronto, Ontario, Canada. Patients were recruited by mediaadvertisement, referral from the MSH outpatient program and from otherclinical centres.

This protocol describes an open pilot study to investigate FormulationA's potential antidepressant activity. The goal of the pilot study wasto demonstrate that Formulation A has the potential to significantlyimprove MDD beyond the levels of the known placebo effect wellestablished in other trials and that Formulation A is an acceptabletreatment in this patient population. Secondary aims of this pilot studywere to evaluate the effect of Formulation A on reducing symptoms ofanxiety and improving quality of life.

Each patient was screened for MDD using DSM-IV TR criteria and theHAM-D. Once entered, they were assigned to the open-label Formulation Astudy for a period of 8 weeks. The patients were further assessed by aglobal measure, the CGI severity (GCI-S) and improvement (CGI-I) scales.Side effects were systematically evaluated using The Udvalg for KliniskeUndersøgelser (UKU) Side Effect Rating Scale (Lingjaerde). Secondarymeasures of depressive symptoms were the Montgomery Asberg DepressionRating Scale (MADRS) and the Beck Depression Inventory (BDI) as selfassessment instruments. The quality of life dimension was assessed withthe Short-Form 36 (SF-36). Anxiety was assessed using the 14 item HAM-A.

In the fixed dose open trial, patients were treated for depression basedon standard treatment protocols for depression. The investigatorsdetermined the severity of depression with the rating scales atbaseline, and at repeated visits at weeks (W) 2, 4, 6, and at week 8. Inintervening weeks patients were seen in brief clinical assessments (V)to evaluate depression and medication tolerance.

Dosage of Formulation A

The dosage of Formulation A was about 2000 mg/day (2 Formulation Acapsules of about 500 mg each, taken orally twice a day).

Inclusion Criteria of Patients

For inclusion in this study, patients had to have met a number ofinclusion criteria, including criteria (i)-(vi), as described below.

-   -   (i) A clinical diagnosis fulfilling DSM-IV TR criteria for major        depressive disorder, single episode or recurrent.    -   (ii) 17-Item Hamilton Depression Rating Scale (HAM-D 17-item)        total score at baseline of 18 or higher.    -   (iii) Males/Females 18-65 years of age who require a new ora        change in their medication treatment for diagnosed major        depression. Treatment decisions were made solely upon the        clinician's judgment of the standard of care appropriate to that        patient. However augmentation strategies are not permitted        during the 8 week trial,    -   (iv) English language literacy.    -   (v) Signed written informed consent obtained.    -   (vi) A negative pregnancy test at screening.

Exclusion Criteria

Patients were excluded from this study if they met a number of exclusioncriteria, including criteria (i)-(xiii), as described below.

-   -   (i) Any other DSM IV TR diagnosis including a clinical diagnosis        of depression other than DSM-IV TR MDD (single        episode/recurrent, e.g., chronic depression and/or refractory        depression were excluded).    -   (ii) Judged to be at significant risk for suicide (HAMD suicide        item >1) or having a history suggesting significant current        potential for self harm.    -   (iii) Any antidepressant medication other than Formulation A.    -   (iv) Subjects who were taking and unable or unwilling to        discontinue natural health products used for depression.    -   (v) Women who were pregnant, breast-feeding, intending to become        pregnant in the next 12 months or on insufficient contraceptive        protection.    -   (vi) Clinically significant organ system diseases, e.g.,        cardiovascular, hepatic, renal, endocrine, gastrointestinal,        metabolic, or other systemic diseases.    -   (vii) Course of electroconvulsive therapy (ECT) during the        observational period.    -   (viii) Suffer from a major neurological condition (i.e.,        Parkinson's disease, Huntington's disease), cerebrovascular        disease (i.e., stroke), metabolic conditions (i.e., Vitamin B12        deficiency), autoimmune conditions (i.e., systematic lupus        erythematosus), viral or other infections (i.e., hepatitis,        mononucleosis, human immunodeficiency), or cancer.    -   (ix) Clinical or subclinical hypo/hyper thyroidism (e.g.,        elevated TSH).    -   (x) Allergies to poultry or eggs.    -   (xi) Subjects who were receiving psychotherapy or who began        psychotherapy during the trial.    -   (xii) Subjects with clinically significant abnormal laboratory        results from screening blood and urinalysis.    -   (xiii) Subjects who became significantly worse during the        washout period.

Study Design

This was a single site, open-label, randomised study of 25 patientsdesigned to validate the efficacy and safety of Formulation, Amonotherapy.

The trial consisted of an 8 week evaluation period preceded if necessaryby a 2 week antidepressant washout period.

Screening

Once the physician and/or research coordinator fully informed thesubject of the study, the nature of the treatment, and the other optionsavailable to them, and the subject signed the informed consent document,the physician made the clinical DSM IV TR diagnosis and administered theHAM-D 17. Eligible subjects then had a medical, psychiatric history andconcomitant therapy review followed by a physical examination. Inaddition, baseline laboratory tests were taken by the researchcoordinator including urine (Routine & Microscopic), CBC differentialand platelets, electrolytes, bilirubin, BUN, creatinine, TSH, LiverFunction Tests, Serum creatinine, and ECG. A pregnancy screen for femalepatients was obtained by hCG blood test. Pregnant patients and thosewith clinically significant abnormal laboratory tests were excluded.

Week 0

Patients returned for a Baseline visit (Week 0) and were assigned toFormulation A monotherapy by the physician. Patients who were depressedand on a current but ineffective antidepressant were offered the switchto Formulation A.

Following Weeks

Following the initial assessment and initiation of Formulation A therapy(V1 and V2) the scheduled visits occurred every week for 8 weeks (W2-W8,V3-V6). Those who were on another antidepressant drug and who chose toenter the study entered a 1-2 week washout period before beginning the 8week active drug trial. The washout period was at the clinicaldiscretion of the physician. During this time, patients were monitoredin a visit one week into the washout by the psychiatrist and furthermonitored by phone by the study coordinator mid-week. It is recognizedthat depression may worsen during the washout period. However, if theprior drug was ineffective or partially ineffective the risks that a 1-2week delay will significantly induce depressive decline in this protocolare not substantially greater than usual care as long as subjects arecarefully monitored during this time and appropriate interventioninstituted as necessary. If Formulation A were not to be an effectiveantidepressant for a particular patient, the patient may be at risk forundue prolongation of depression. However, depression is a chronicdisorder which is generally present for months prior to being diagnosedor treated so an additional 8 weeks in the presence of carefulmonitoring together with institution of Formulation A, a potentiallyeffective medication, should not be substantially different fromstandard care. Moreover, standard care, as already discussed, is onlyeffective in about 60% of patients and therefore often requires the samepossible reevaluations and drug alterations.

At V2 (may be combined with V1 (WO)) to V6 (W8), the followingprocedures were performed by the supervising psychiatrist (PI) and/orthe research coordinator:

-   -   Weight    -   Height    -   Vital Signs    -   Hamilton Depression Rating Scale (17 item) (HAM-D 17) (Hamilton        1967).    -   Clinical Global Impression (CGI-S, CGI-I) (Guy)    -   Montgomery-Asberg Depression Rating Scale (MADRS) (Montgomery)    -   Beck Depression Inventory Scale (BDI) (10).—Quality of Life        (SF-36) (Ware    -   Hamilton rating scale for anxiety (HAMA) (Hamilton 1959)    -   Udvalg for Kliniske Undersogelser (UKU) (Lingjaerde) (Reporting        of Adverse Events) (except at V2)    -   Medication Compliance (except at V2)

Study visits were estimated at about one hour with the exception of thebaseline visit which may have taken 2 hours.

If subjects became more depressed while in the study they were evaluatedby the principal investigator to determine the best clinical approach.If deemed necessary, Formulation A was stopped in favour of anotherantidepressant treatment. This was a clinical decision made solely onthe basis of best practices in the treatment of depression and on thepatient's best clinical interests.

General supportive contact with the patients by the physician and theresearch coordinator was permitted, and the contact was generallyrestricted to answering pertinent questions about the patient's illnesscourse and treatment. No formal psychotherapy was permitted.

Statistical Methods.

The primary effect was tested using a repeated analysis of variance withthe scores of the HAM-D 17 as the outcome variable. A significant timeeffect supports the hypothesis. The total anticipated sample size of 25patients was large enough to detect changes on the HAM-D 17 as follows0.65 standard deviations (two-tailed one sample P<0.05). The reportedstandard deviations on the HAM-D-17 were in the range of 4.5 to 6.5.Therefore, the design of this study had 80% power to detect averagechanges as small as 4.3 points on this 52 point scale. As per inclusioncriteria, participants each had a HAM-D 17 score of greater than 17. TheFranck criterion for remission was a HAM-D 17 of 9 or under. This studyused a more conservative and accepted level of 7 or under. The effectsize of 4.3 was sufficiently sensitive to detect clinical improvementfrom scores greater than 17 to scores less than 10. Positive outcome wasstatistically based on an expected placebo response rate ranging from30% to 50% in treatment trials for depression. In this study, a placeboresponse rate of 40% was assumed. Some analysis of responders andremitters was carried out as appropriate.

Results

A total of 23 patients were entered into the study. Three of thesubjects, (#104, #105 and #118) were never treated and hence, theirresults were not considered analyzable. Of the 20 subjects that receivedat least one dose of Formulation A, 16 of them completed the 8 weekstudy. The remaining 4 subjects did not complete the 8 week study, butsince they each received at least one dose of Formulation A, theirresults were deemed analyzable. The reasons these 4 subjects did notcomplete the entire study included non-compliance with the medicationsand/or appointments, impatience with the results, and the subjectleaving the country.

The results for the 20 subjects who received at least one dose ofFormulation A are provided in the below tables.

Total Score Sheet for HAM-D Screen Week 0 Week 2 Week 4 Visit 7 Week 6Week 8 Subject #101 20 14 11 7 3 Subject # 102 19 15 5 2 0 Subject # 10322 7 3 5 0 Subject # 106 21 4 8 10 12 Subject # 107 22 17 20 Subject #108 20 14 19 Subject # 109 25 16 17 20 24 Subject # 110 21 10 17 8 4Subject # 111 24 20 19 19 23 23 Subject # 112 29 8 5 2 0 Subject # 11333 13 9 11 8 Subject # 114 29 13 19 22 30 Subject # 115 32 8 13 5 6Subject # 116 19 17 24 17 24 Subject # 117 23 11 9 8 Subject # 119 23 2320 13 10 Subject # 120 23 5 Subject # 121 23 11 8 6 3 Subject # 122 3222 16 23 16 Subject # 123 24 19 12 10 11

Total Score Sheet for GHQ Screen Week 0 Week 2 Week 4 Visit 7 Week 6Week 8 Subject #101 15 27 14 11 3 Subject # 102 22 11 3 0 0 Subject #103 18 9 13 2 2 Subject # 106 22 10 10 6 10 Subject # 107 27 12 9Subject # 108 27 19 17 Subject # 109 25 16 16 17 20 Subject # 110 28 1316 9 8 Subject # 111 26 15 20 21 21 20 Subject # 112 30 15 9 4 0 Subject# 113 34 13 12 5 2 Subject # 114 33 25 22 22 28 Subject # 115 31 2 7 5 8Subject # 116 32 26 25 25 24 Subject # 117 24 14 7 8 Subject # 119 31 1927 20 8 Subject # 120 23 8 Subject # 121 35 7 1 1 1 Subject # 122 31 2310 23 15 Subject # 123 26 17 11 4 2

Total Score Sheet for MADRS Screen Week 0 Week 2 Week 4 Visit 7 Week 6Week 8 Subject #101 34 24 28 10 6 Subject # 102 30 18 10 0 2 Subject #103 28 10 4 2 2 Subject # 106 30 14 14 18 24 Subject # 107 38 28 28Subject # 108 20 18 22 Subject # 109 28 23 20 28 26 Subject # 110 28 1636 14 10 Subject # 111 46 40 40 40 32 36 Subject # 112 38 16 10 10 2Subject # 113 46 16 18 14 6 Subject # 114 42 26 38 38 44 Subject # 11532 12 12 10 10 Subject # 116 36 42 44 34 46 Subject # 117 36 22 10 6Subject # 119 38 34 34 18 10 Subject # 120 32 6 Subject # 121 38 14 6 64 Subject # 122 44 38 22 32 24 Subject # 123 30 28 22 14 16

Total Score Sheet for BDI-21 Screen Week 0 Week 2 Week 4 Visit 7 Week 6Week 8 Subject #101 27 25 27 17 10 Subject # 102 25 13 6 0 1 Subject #103 26 14 10 8 8 Subject # 106 30 12 7 12 40 Subject # 107 33 28 26Subject # 108 32 14 20 Subject # 109 29 23 24 20 25 Subject # 110 29 2224 13 8 Subject # 111 32 28 27 33 27 27 Subject # 112 37 21 10 9 1Subject # 113 53 23 22 18 3 Subject # 114 54 40 52 52 59 Subject # 11539 13 16 3 4 Subject # 116 38 37 37 42 40 Subject # 117 24 20 11 7Subject # 119 35 36 40 24 18 Subject # 120 26 1 Subject # 121 43 10 4 33 Subject # 122 55 38 25 46 27 Subject # 123 33 34 22 5 10

Total Score Sheet for HAM-A Screen Week 0 Week 2 Week 4 Visit 7 Week 6Week 8 Subject #101 15 9 8 7 3 Subject # 102 13 8 2 0 1 Subject # 103 87 2 0 2 Subject # 106 21 5 5 5 10 Subject # 107 12 7 13 Subject # 108 158 11 Subject # 109 17 12 13 18 14 Subject # 110 14 8 9 2 5 Subject # 11133 29 24 32 23 24 Subject # 112 22 10 4 1 2 Subject # 113 35 22 14 4 3Subject # 114 24 10 18 21 21 Subject # 115 25 10 12 4 5 Subject # 116 1213 15 12 16 Subject # 117 21 10 8 7 Subject # 119 13 19 15 13 6 Subject# 120 14 4 Subject # 121 24 9 3 4 2 Subject # 122 43 31 21 32 22 Subject# 123 23 27 14 8 10

Total Score Sheet for ASEX Screen Week 0 Week 2 Week 4 Visit 7 Week 6Week 8 Subject #101 12 14 12 13 13 Subject # 102 21 23 19 19 15 Subject# 103 12 12 17 17 13 Subject # 106 10 9 7 7 10 Subject # 107 15 14 16Subject # 108 15 11 12 Subject # 109 17 15 17 19 19 Subject # 110 26 2528 28 27 Subject # 111 28 28 28 28 28 28 Subject # 112 11 13 11 11 9Subject # 113 28 28 28 28 9 Subject # 114 28 28 28 30 30 Subject # 11519 17 17 11 15 Subject # 116 11 10 11 12 12 Subject # 117 14 12 18 16Subject # 119 30 30 28 30 30 Subject # 120 19 15 Subject # 121 18 26 1222 10 Subject # 122 26 14 15 30 12 Subject # 123 22 20 20 16 20

Response Rate and Intensity of Response

The following definitions were used to assess each subject's response totreatment with Formulation A. A “responder” or “ever-responder” is asubject with at least 50% improvement on the Hamilton Depression RatingScale (HAM-D score) as compared to baseline score at any time during thestudy. A “clinical responder” is a subject meeting the “responder”criteria who, in the opinion of the Principal Investigator has apositive clinical outcome. An “end of study responder” is a subjectmeeting response criteria at the end of the study (or at lastobservation). “Remission” is a reduction of the HAM-D score to less than8.

The above study showed that of the 20 subjects who received at least onedose of Formulations A, 15 of them (75%) were ever-responders, and 14 ofthem (70%) were clinical responders. In addition, among the 16 subjectswho completed the 8 week study, the number of ever-responders was 13/16(81.3%) and the number of clinical responders was 12/16 (75%). Inaddition, of the 16 subjects who completed the study, the overall dropin HAM-D score (including non-responders) was significant at 56.08%. Thedrop in HAM-D score among the ever-responders who completed the 8 weekstudy was higher, at 68.1%, a figure well beyond the minimal 50% droprate require for an ever-response.

Note should be made of two subjects whose response was influenced byenvironmental circumstances. Subject #114, who was not included amongthe clinical responders, was responsive by week 2 when her HAM-D scorefell by more than 50% on Formulation A; but external factors intervened.She began to encounter medical problems (not related to Formulation A)and difficulty at work when she applied for disability insurance. Theseenvironmental factors completely overtook her good emotional response toFormulation A.

Based on the strict criterion of a 50% reduction in the HAM-D score,subject #106 would not have been considered a responder at week 8because at that time her score was 12, just short of a 50% drop from herentry score of 21. Throughout the 8 week trial, however, subject #106did respond with scores of 4 (week 2), 8 (week 4) and 10 (week 6) Infact, during the study, subject #106 was deemed to be a clinicalresponder by the PI and was entered into the Extension Study (seeExample 7) where scores of 1, 11, 7 and 9 were recorded. After startingthe Extension Study, subject #106 was faced with considerable familyturmoil which disrupted her positive response to Formulation A. Whenthis turmoil subsided she continued to maintain responsiveness toFormulation A. No medication can completely offset the traumatic effectsof environmental circumstances. Formulation A may well have amelioratedthe emotional trauma of those circumstances for subject #106.

Remission Rate

Not all ever-responders went into remission and not everyone who wentinto remission remained there until the end of the 8 week study. Nine ofthe 15 ever-responders (60%) went into remission at some point duringthe 8 week study. Seven of those nine subjects (77.8%; or 46.7% of allstudy participants) who achieved remission remained in remission by theend of the 8 week study.

The table below delineates all those study participants who went intoremission and all who stayed in remission. Checkmarks indicate that thesubject went into remission or had sustained remission, while X marksindicate that the subject did not go into remission or did not havesustained remission to week 8 of the study.

Remission at any Remission Sustained to 8 Subject time Weeks 101 ✓ ✓ 102✓ ✓ 103 ✓ ✓ 106 ✓ X 110 ✓ ✓ 112 ✓ ✓ 113 X X 114 X X 115 ✓ ✓ 117 X X 119X X 120 ✓ withdrew 121 ✓ ✓ 122 X X 123 X X N ever in N in sustainedremission = 7 remission = 9 (46.7%) (60%)

In addition, a major secondary outcome, reduction in anxiety, wasexperienced by all of the ever-responders except one. These results showthat Formulation A is effective in the treatment of major depressivedisorder and anxiety. Furthermore, there were no serious side-effectsattributable to the drug. There was no increase in weight, nor was therea diminution in sexual function in subjects participating in the study.

Example 7

The positive efficacy and safety results of the study described inExample 6 necessitated an Extension Study. Ten subjects from the studydescribed in Example 6 were entered into the Extension Study. TheExtension Study was open only to those subjects from the study describedin Example 6 who were clinical responders at the end of that 8 weekstudy. Formulation A was administered as described in Example 6 and thesubjects in the Extension Study were analyzed on a monthly basis for 10months. The below table show the HAM-D scores of the subjects in theExtension Study.

Subject Subject Subject Subject Subject Subject Subject Subject SubjectSubject Month # 102 # 103 # 106 # 110 # 112 # 113 # 115 # 119 # 121 #123 Visit 1 0 0 12 4 0 8 6 10 3 11 Visit 2 0 2 1 5 2 9 2 10 4 14 Visit 41 8 11 3 1 10 w/d 3 5 14 Visit 4 0 2 7 1 1 18 10 1 3 Visit 5 0 3 9 2 0w/d 4 5 Visit 6 0 1 16 2 1 w/d Visit 7 0 w/d 9 2 0 Visit 8 1 6 4 0 Visit9 1 11 1 Visit 10 2 w/d = withdrawn from Extension Study

Four of the 10 subjects were withdrawn from the Extension Study due tooccurrence of an exclusionary criterion for continuing in the study. Theresults of this Extension Study showed that all of the subjects in thestudy were, by definition, responders to Formulation A. Six of the 10clinical responders (60%) were in remission at the outset of theExtension Study. Eight of the 10 subjects (80%) were in remission at thelast date of assessment. Two of the subjects were clinical responders inthe initial 8 week study but had not gone into remission until in theExtension Study. Only one subject (#113) who entered the Extension Studyas a clinical responder relapsed after entering the Extension Study. Thepositive efficacy and safety results of the study described in Example 6and the Extension Study has necessitated a further Extension Study.

Example 8

As described in the above Examples, Formulation A has a demonstratedtherapeutic action. Studies have been conducted to investigate themechanism of action of Formulation A. In particular, studies wereconducted to determine inhibition of binding interactions betweenradioligands and their receptors, or inhibition of radio-labeled enzymesto act on their associated target proteins by Formulation A. The levelof inhibition by Formulation A (measured as percent inhibition ofspecific binding to each receptor by Formulation A) was determined. Thetesting of inhibition of binding interactions and enzymatic activitieswas performed in duplicate for each sample at two differentconcentrations of Formulation A (1.0 μg/mL and 10.0 μg/mL). Theseconcentrations of Formulation A were prepared by dissolving the contentsof a capsule of Formulation A in dimethyl sulfoxide and subsequentlydiluting the solution to either 1.0 μg/mL or 10.0 μg/mL of FormulationA. These diluted solutions were called Isolate A. Radioligand bindingassays were then performed using the receptors and enzymes listed in thebelow table. The average percent inhibition of specific binding at eachconcentration of Isolate A was determined. The results of this study areshown in the below table.

Percent Inhibition Percent Inhibition at 1.0 μg/mL of at 10.0 μg/mLReceptor/Enzyme Isolate A of Isolate A Neurotransmitter Related:Adenosine, Non-selective 4.52 12.06 Adrenergic, Alpha 1, Non-selective−5.01 −0.56 Adrenergic, Alpha 2, Non-selective 0.52 7.88 Adrenergic,Beta, Non-selective 3.97 17.38 Dopamine Transporter 4.73 −1.61 Dopamine,Non-selective −11.52 −0.59 GABA A, Agonist Site 1.11 10.58 GABA A, BDZ,alpha 1 site 3.18 11.96 GABA-B 19.85 14.33 Glutamate, AMPA Site 5.7929.05 (Ionotropic) Glutamate, Kainate Site (Iontropic) 3.96 22.38Glutamate, NMDA, Agonist Site 1.12 34.59 (Ionotropic) Glutamate, NMDA,Glycine 5.63 27.45 (Strychnine-insensitive site) (Ionotropic) Glycine,strychnine-sensitive 7.29 8.86 Histamine, H1 −11.22 −8.21 Histamine, H2−0.47 3.83 Histamine, H3 14.37 −6.79 Melatonin, Non-selective −12.91−3.64 Muscarinic, M1 (hr) −4.72 −17.33 Muscarinic, M2 (h) 1.52 1.19Muscarinic, Non-selective, Central −12.00 −8.66 Muscarinic,Non-selective, −11.85 −9.74 Perhpheral Nicotinic, Neuronal (a-BnTx −0.73−1.74 insensitive) Norepinephrine Transporter 6.12 7.68 Opioid,Non-selective 9.10 7.72 Orphanin, ORL1 (h) −11.04 −10.55 SerotoninTransporter −0.81 5.46 Serotonin, Non-selective −1.24 5.06 Sigma,Non-selective 0.38 0.60 Steroids: Estrogen 2.23 7.46 Testosterone(cytosolic) (h) 5.80 6.16 Ion Channels: Calcium Channel, Type L 0.17−6.09 (Dihydropyridine site) Calcium Channel, Type N 5.01 5.02 PotassiumChannel, ATP-Sensitive −1.00 2.46 Potassium Channel, Ca²⁺ Act., VI −0.72−1.23 Potassium Channel, I [Kr] (hERG) −12.25 −10.99 (h) Sodium, Site 2−0.73 9.17 Second Messengers: Nitric Oxide, NOS (Neuronal- −19.63 −0.04Binding) Prostaglandins: Leukotriene, LTB4 (BLT) −0.39 −8.11Leukotriene, LTD4 (CysLT1) 14.10 19.68 Thromboxane A2 (h) −2.41 5.28Growth Factors/Hormones: Corticotropin Releasing Factor, −4.31 8.08Non-selective Oxytocin 14.83 17.68 Platelet Activating Factor, PAF−15.15 5.99 Thyrotropin Releasing Hormone, 3.59 7.53 TRH Brain/GutPeptides: Angiotensin II, AT1 (h) −10.80 −8.76 Angiotensin II, AT2 8.8210.55 Bradykinin, BK2 −18.67 −6.65 Cholecystokinin, CCK1 (CCKA) −20.25−7.09 Cholecystokinin, CCK2 (CCKB) −0.31 −1.74 Endothelin, ET-A (h)15.33 −9.23 Endothelin, ET-B (h) 19.44 26.76 Galanin, Non-selective 7.9420.49 Neurokinin, NK1 19.32 13.63 Neurokinin, NK2, (NKA) (h) 15.62 32.15Neurokinin, NK3 (NKB) −7.70 1.09 Vasoactive Intestinal Peptide, Non-18.20 −1.65 selective Vasopressin 1 8.63 1.84 Enzymes: Decarboxylase,Glutamic Acid 9.11 5.63 Esterase, Acetylcholine −2.21 −1.78 Oxidase,MAO-A, Peripheral −11.09 −3.83 Oxidase, MOA-B, Peripheral −18.26 −15.82Transferase, Choline Acetyl 18.06 20.00

In general, a binding inhibition or an enzymatic activity inhibition of20% or less is indicative that a given test compound is inactive at thatparticular receptor binding site or target protein. A binding inhibitionor an enzymatic activity inhibition of more than 20% is indicative thatthe test compound has activity at that particular binding site or enzymesite. The above-described binding inhibition studies showed that IsolateA displaced glutamate from four of its major ionotropic receptors.Binding by radio-labeled AMPA to the AMPA receptor was inhibited by29.05% in the presence of Isolate A (10 μg/mL). Binding by radio-labeledkainic acid to the kainate receptor was inhibited by 22.38% in thepresence of Isolate A (10 μg/mL). Binding by radio-labeled CGP 39653 tothe agonist site of the NMDA receptor was inhibited by 34.59% in thepresence of Isolate A (10 μg/mL). Binding by radio-labeled MDL-105,519to the glycine site that is strychnine-insensitive of the NMDA receptorwas inhibited by 27.45% in the presence of Isolate A (10 μg/mL). Inaddition, binding by neurokinin A to the NK2 receptor was inhibited by32.15% in the presence of Isolate A (10 μg/mL). Additional detailsregarding the binding inhibition experiments for each of the five abovedescribed receptors are provided in the following table.

K_(i) of the Reference Reference Compound Receptor Radioligand K_(d) (M)Compound (M) Glutamate, [³H]AMPA 2.8 × 10⁻⁸ (+/−) AMPA 2.64 × 10⁻⁸ AMPAHBr Glutamate [³H]Kainic 1.6 × 10⁻⁸ Kainic Acid 1.05 × 10⁻⁸ Kainate AcidGlutamate, [³H]CGP   7 × 10⁻⁹ NMDA 9.63 × 10⁻⁶ NMDA 39653 Agonist siteGlutamate, [³H]-MDL-   2 × 10⁻⁸ MDL- 1.72 × 10⁻⁸ NMDA, 105,519 105,519glycine (strychnine- insensitive site) Neurokinin 2 [¹²⁵I]-  5 × 10⁻¹⁰Neurokinin  2.53 × 10⁻¹⁰ (NK2) Neurokinin A A

The ionotropic glutamate receptors, the NK2 receptor and the neurokinin1 (NK1) receptor were used in additional receptor binding assays. Aone-concentration controlled experiment was performed to assess theability of various isolates of the contents of a Formulation A capsuleto antagonize ligand binding by various glutamate receptors and the NK2receptor. The AMPA receptor, kainate receptor, the agonist and glycine(strychnine-insensitive) binding sites of the NMDA receptor, as well asthe NK2 receptor were studied in this assay. The contents of FormulationA capsules were dissolved using various solvents and extracted usingfour different processes, as described in detail below. These extractionprocedures resulted in a number of fertilized egg isolates. Theseisolates were called: Sample #19 Top Isolate, Sample #19 Bottom Isolate,Sample #20 Top Isolate, Sample #20 Bottom Isolate, Fraction X Isolateand Sample #2 Isolate. These isolates were then each tested in theradioligand binding assay.

Sample #19 was prepared by weighing out 103 mg of the contents of aFormulation A capsule. Water (10.3 mL) was added and the solution wasvortexed for one minute. Thirty mL of ethyl acetate was then added tothe solution and the solution was vortexed again for 1 minute. Thesample was then centrifuged using a bench top Beckman centrifuge. Threefractions were formed as a result. The top (organic) and bottom(aqueous) fractions were collected separately and the middle fractionwas discarded. The top and bottom fractions were each dried down. Thebottom (aqueous) fraction was reconstituted in 2.06 mL of water. Thesample was not clear and it was centrifuged at 10,000 rpm for tenminutes using a microcentrifuge. The supernatant was removed, labeled assample 085426-4 (Sample #19 Bottom Isolate) and used in the receptorbinding studies. The top (organic) fraction was reconstituted in 1.245mL of 20% acetonitrile in water. The sample was not clear and it wascentrifuged at 10,000 rpm for ten minutes using a microcentrifuge. Thesupernatant was removed, labeled as sample 085426-3 (Sample #19 TopIsolate) and used in the receptor binding studies. A control for sample#19 was also made. This control consisted of 20% acetonitrile in water,and was labeled as sample 085426-5 in the receptor binding studies.

Sample #20 was prepared by weighing out 249.7 mg of the contents of aFormulation A capsule. Ten mL of 1:1 methanol:dichloromethane was addedand the solution was vortexed. Ten mL of dichloromethane was then addedto the solution and the solution was vortexed again. The sample was thencentrifuged at 3500 rpm for fifteen minutes using a bench-top Beckmancentrifuge. Three fractions were formed as a result. The top and bottomorganic fractions were collected separately. The middle fraction wasdiscarded. The top and bottom fractions were each dried down andreconstituted in 2.49 mL of 100% methanol in water. The top methanolfraction was semi-clear and the bottom dichloromethane fraction was notsoluble. Both samples were centrifuged at 10,000 rpm for ten minutesusing a microcentrifuge. The supernatant of each sample was removed. Thesupernatant from the top methanol fraction was labeled as sample085426-6 (Sample #20 Top Isolate) and used in the receptor bindingstudies. The supernatant for the bottom dichloromethane fraction waslabeled as sample 085426-7 (Sample #20 Bottom Isolate) and used in thereceptor binding studies. A control for sample #20 was also made. Thiscontrol consisted of 10% methanol in water, and was labeled as sample085426-9 in the receptor binding studies.

Sample Fraction X was prepared as follows. One hundred twenty-one mg ofthe contents of a Formulation A capsule was weighed out. Ten mL of waterwas then added. Ten mL of dichloromethane was then added to the solutionand the sample was vortexed. The aqueous and organic fractions were eachseparately removed. The solvent isolation was repeated by adding 10 mLof dichloromethane to the aqueous fraction and vortexing the solution.Again, the aqueous and organic fractions were each separately removed.The organic fractions from the two isolations were combined and theaqueous fractions from the two isolations were combined. The aqueous andorganic fractions were dried down and weighed. The aqueous fractionweighed 116.4 mg and the organic fraction weighed 1.3 mg. The organicfraction was reconstituted in 1.3 mL of 10% methanol in water (for aconcentration of 0.1 mg/mL), labeled as sample 085426-8 (Fraction XIsolate) and used in the binding studies. A control for sample FractionX was also made. This control consisted of 10% methanol in water, andwas labeled as sample 085426-9 (note this was the same control as usedfor sample #20) in the receptor binding studies.

Sample #2 was prepared as follows. A portion (1.8 mg) of the contents ofa Formulation A capsule was weighed out. Forty percent PEG in water plus0.25% Tween 80 (3.6 mL) was then added (for a concentration of 0.5mg/mL) and the sample was vortexed. This preparation was labeled assample 085426-1 (Sample #2 Isolate) and was tested in the receptorbinding studies. A control for sample #2 was also made. This controlconsisted of 40% PEG in water plus 0.25% Tween 80, and was labeled assample 085426-2 in the receptor binding studies.

The results (obtained from duplicate samples of each isolate at maximalconcentrations) from the receptor binding study are presented in thefollowing table.

# 19 #19 #20 #20 #20/ Target Top Bottom #19 Fraction X Top BottomFraction X #2 #2 Receptor Isolate Isolate Control Isolate IsolateIsolate Control Isolate Control Glutamate, 6% 95% 3% −10% 97% 0% 14% 38%−5% AMPA Glutamate, −9% 100% −22% 1% 101% 6% 2% 21% −5% KainateGlutamate, 2% 106% −6% −10% 110% −10% 0% 49% −23% NMDA, AgonistGlutamate, −6% 63% 4% −12% 92% −34% −19% −18% −56% NMDA, glycineNeurokinin 1 24% 9% 33% 25% 21% −11% 12% 87% 86% Neurokinin 2 18% 13%12% 55% 53% 18% 1% 102% 102% Bolded data indicate more than 50%inhibition at the concentration tested.

Binding by radio-labeled AMPA to the AMPA receptor was inhibited by 97%in the presence of Sample #20 Top Isolate. Binding by radio-labeledkainic acid to the kainate receptor was inhibited by 101% in thepresence of Sample #20 Top Isolate. Binding by radio-labeled CGP 39653to the agonist site of the NMDA receptor was inhibited by 110% in thepresence of Sample #20 Top Isolate. Binding by radio-labeled MDL-105,519to the glycine site that is strychnine-insensitive of the NMDA receptorwas inhibited by 92% in the presence of Sample #20 Top Isolate. Inaddition, the Fraction X Isolate inhibited neurokinin A binding to itsNK2 receptor by 55%. To the knowledge of the inventors, this is thefirst instance in which glutamate receptors and the NK2 receptor havebeen shown to be antagonized by a single substance.

To further confirm that binding to and activation of the glutamatereceptors discussed above and the NK2 receptor are antagonized by Sample#20 Top Isolate, prepared as described above, dose response studies wereconducted. Inhibition of binding of the AMPA receptor, the kainatereceptor, the agonist site of the NMDA receptor, the glycine(strychnine-insensitive site) of the NMDA receptor and the NK2 receptorwere evaluated in the presence of the following concentrations of #20Top Isolate (based on the amount of Formulation A contained in Sample#20 prior to extraction): 0.1, 0.3, 1.0, 3.0, 10, 30, 100 and 300 μg/mL.

FIG. 8 shows the results of the assay performed with the agonist site(ionotropic) of the NMDA receptor. Sample #20 Top Isolate inhibited theability of CGP 39653 to bind the agonist site of the NMDA receptor in aconcentration-dependent manner, with higher concentrations of Sample #20Top Isolate providing more binding inhibition and lower concentrationsproviding less binding inhibition. The IC₅₀ of NMDA was determined to be1.11×10⁻⁵ μg/mL and the K_(i) was determined to be 9.02×10⁻⁶ M. The IC₅₀of Sample #20 top Isolate was determined to be 5.78 μg/mL and the K_(i)was determined to be 4.69 M.

FIG. 9 shows the results of the assay performed with the kainatereceptor. Sample #20 Top Isolate inhibited the ability of glutamatekainic acid to bind the kainate receptor in a concentration-dependentmanner, with higher concentrations of Sample #20 Top Isolate providingmore binding inhibition and lower concentrations providing less bindinginhibition. The IC₅₀ of kainic acid was determined to be 1.77×10⁻⁸ μg/mLand the K_(i) was determined to be 1.08×10⁻⁸ M. The IC₅₀ of Sample #20Top Isolate was determined to be 12.0 μg/mL and the K_(i) was determinedto be 7.32 M.

FIG. 10 shows the results of the assay performed with the AMPA receptor.Sample #20 Top Isolate inhibited the ability of AMPA to bind the AMPAreceptor in a concentration-dependent manner, with higher concentrationsof Sample #20 Top Isolate providing more binding inhibition and lowerconcentrations providing less binding inhibition. The IC₅₀ of (+/−) AMPAHBr was determined to be 3.54×10⁻⁸ μg/mL and the K_(i) was determined tobe 3.00×10⁻⁸ M. The IC₅₀ of Sample #20 Top Isolate was determined to be11.7 μg/mL and the K_(i) was determined to be 9.91 M.

FIG. 11 shows the results of the assay performed with the glycine(strychnine-insensitive) site of the NMDA receptor. Sample #20 TopIsolate inhibited the ability of MDL-105,519 to bind the glycine(strychnine-insensitive) site of the NMDA receptor in aconcentration-dependent manner, with higher concentrations of Sample #20Top Isolate providing more binding inhibition and lower concentrationsproviding less binding inhibition. The IC₅₀ of MDL-105,519 wasdetermined to be 2.8×10⁻⁸ μg/mL and the K_(i) was determined to be2.36×10⁻⁸ M. The IC₅₀ of Sample #20 Top Isolate was determined to be21.9 μg/mL and the K_(i) was determined to be 18.4 M.

FIG. 12 shows the results of the assay performed with the NK2 receptor.Sample #20 Top Isolate inhibited the ability of neurokinin A to bind theNK2 receptor in a concentration-dependent manner, with higherconcentrations of Sample #20 Top Isolate providing more bindinginhibition and lower concentrations providing less binding inhibition.The IC₅₀ of neurokinin A was determined to be 6.84×10⁻¹° μg/mL and theK_(i) was determined to be 5.76×10⁻¹° M. The IC₅₀ of Sample #20 TopIsolate was determined to be 4.15×10² μg/mL and the K_(i) was determinedto be 3.49×10² M.

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What is claimed is:
 1. A method for treating or preventing a disorderusing fertilized egg isolate, said disorder being selected from thegroup consisting of major depressive mood disorders and anxietydisorders, and said method comprising the step of administering atherapeutically effective amount of the fertilized egg isolate to apatient in need thereof.
 2. The method according to claim 1, wherein thefertilized egg isolate comprises freeze-dried embryo, ovalbumin, andclear sac from the fertilized egg.
 3. The method according to claim 1,wherein the disorder is major depressive disorder.
 4. The methodaccording to claim 1, wherein the disorder is anxiety.
 5. The methodaccording to claim 1, wherein the fertilized egg isolate is an aqueousisolate produced by the process comprising the steps of (i)substantially separating the contents of at least one fertilized eggfrom the shell; (ii) optionally filtering the contents to produce aretentate; (iii) combining the contents or retentate in a container;(iv) blending, optionally with solvent added, the contents or retentateto produce a slurry; (v) mixing the slurry with an aqueous solution fora period of time; (vi) clarifying the mixture to produce an aqueoussolution substantially free of solid material; (vii) separating theaqueous solution from the solid material, (viii) freezing the aqueoussolution; and (ix) freeze-drying the aqueous solution to produce thefertilized egg isolate.
 6. The method according to claim 5, wherein thedisorder is major depressive disorder.
 7. The method according to claim5, wherein the disorder is anxiety.
 8. The method according to claim 1,wherein the fertilized egg isolate is a solvent isolate produced by theprocess comprising the steps of (i) substantially separating thecontents of at least one fertilized egg from the shell; (ii) optionallyfiltering the contents to produce a retentate; (iii) combining thecontents or retentate in a container; (iv) blending, optionally withsolvent added, the contents or retentate to produce a slurry; (v) mixingthe slurry with a hydrophobic solvent for a period of time; (vi)clarifying the mixture to produce a solution substantially free of solidmaterial; (vii) separating the solution from the solid material; (viii)separating the hydrophobic solvent portion from any aqueous solventportion; and (ix) concentrating the hydrophobic solvent portion toproduce fertilized egg solvent isolate.
 9. The method according to claim8, wherein the disorder is major depressive disorder.
 10. The methodaccording to claim 8, wherein the disorder is anxiety.
 11. A method fortreating or preventing a disease or condition associated with aglutamate receptor using fertilized egg isolate, said method comprisingthe step of administering a therapeutically effective amount of thefertilized egg isolate to a patient in need thereof.
 12. The method ofclaim 11, wherein said glutamate receptor is an AMPA receptor, a kainatereceptor or an NMDA receptor.
 13. The method of claim 12, wherein saiddisease or condition associated with said glutamate receptor isdepression, major depressive disorder, anxiety, Alzheimer's disease,epilepsy, schizophrenia, impairment of brain cell function afterstroke/ischemia, amyotrophic lateral sclerosis (Lou Gehrig's Disease),lathyrism, autism, mental retardation, dementia, disturbances ofcognition, bipolar depression or mania.
 14. The method of claim 13,wherein said disease or condition is major depressive disorder.
 15. Themethod according to claim 13, wherein the fertilized egg isolatecomprises freeze-dried embryo, ovalbumin, and clear sac from thefertilized egg.
 16. The method of claim 15, wherein said disease orcondition is major depressive disorder.
 17. A method for treating orpreventing a disease or condition associated with a neurokinin 2 (NK2)receptor using fertilized egg isolate, said method comprising the stepof administering a therapeutically effective amount of the fertilizedegg isolate to a patient in need thereof.
 18. The method of claim 17,wherein said disease or condition associated with said NK2 receptor isdepression, anxiety, irritable or inflammatory bowel syndrome,inflammatory airway diseases or urinary incontinence.
 19. The method ofclaim 18, wherein said disease or condition is major depressivedisorder.
 20. The method according to claim 18, wherein the fertilizedegg isolate comprises freeze-dried embryo, ovalbumin, and clear sac fromthe fertilized egg.
 21. The method according to claim 1, wherein thefertilized egg isolate does not comprise a substantial amount of theyolk from the fertilized egg.
 22. The method according to claim 1,wherein the patient is not treated by psychotherapy concurrently withthe treatment.
 23. The method according to claim 1, wherein the patientis treated by psychotherapy concurrently with the treatment.